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Fengycin is a lipopeptide antibiotic synthesized nonribosomally by five fengycin synthetases. These enzymes are linked in a specific order to form the complex. This study investigates how these enzymes interact in the complex and analyzes the regions in the enzymes that are critical to the interactions.

ABSTRACT1,2,3,4,6-Penta-O-galloyl-β-d-glucopyranose (PGG) is an active ingredient in plants that are commonly used in Chinese medicine to treat inflammation. We demonstrate here that PGG, at 6.25 μM, does not inhibit the growth of Staphylococcus aureus, and yet it prevents biofilm formation on polystyrene and polycarbonate surfaces. At the same concentration, PGG is not toxic to human epithelial and fibroblast cells. PGG has an IB50value, i.e., the PGG concentration that inhibits 50% biofilm formation, of 3.6 μM. The value is substantially lower than that of N-acetylcysteine, iodoacetamide, and N-phenyl maleimide, which are known to inhibit biofilm formation by S. aureus. Biochemical and scanning electron microscopy results also reveal that PGG inhibits initial attachment of the bacteria to solid surface and the synthesis of polysaccharide intercellular adhesin, explaining how PGG inhibits biofilm formation. The results of this study demonstrate that coating PGG on polystyrene and silicon rubber surfaces with polyaniline prevents biofilm formation, indicating that PGG is highly promising for clinical use in preventing biofilm formation by S. aureus.

ABSTRACTZta, a transcription factor encoded by Epstein-Barr virus, is efficiently translated from a BRLF1-BZLF1 bicistronic mRNA. In this study, we demonstrate that inserting a stem-loop structure, which is known to block ribosome scanning, in the 5' region of the intercistronic region does not prevent the translation of a luciferase reporter protein from the bicistronic mRNA fused with the firefly luciferase gene, suggesting that the translation does not involve translation reinitiation. Mutational analyses reveal that the region between nucleotides 86 and 125 (region I) of the intercistronic region is essential for the translation. Meanwhile, the region between nucleotides 126 and 165 (region II) is also important since, without this region, the translation is inefficient. The region I sequence is partially complementary to the sequence between nucleotides 1489 and 1524 of 18S rRNA. This homology is significant, since disrupting the homology reduces the translation efficiency. Furthermore, luciferase is efficiently translated if the entire intercistronic region is replaced with a sequence complementary to the region between nucleotides 1401 and 1560 of the 18S rRNA. We hypothesize that Rta may assist 40S ribosome in recognizing the region I sequence to start a scanning process for Zta translation.

ABSTRACTRecently, we found that more than 10% of the cases of acute non-A, non-B, non-C hepatitis in Taiwan were caused by a novel strain of hepatitis E virus (HEV). Since none of these patients had a history of travel to areas where HEV is endemic, the source of transmission remains unclear. The recent discovery of a swine HEV in herd pigs in the United States has led us to speculate that HEV may also circulate in herd pigs in Taiwan and may serve as a reservoir for HEV in Taiwan. Of 275 herd pigs obtained from 10 pig farms in Taiwan, 102 (37%) were seropositive for serum anti-HEV immunoglobulin G (IgG). A 185-bp genomic sequence within the ORF-2 of the HEV genome was amplified and cloned from serum samples of an anti-HEV positive pig and subsequently from serum samples of a patient with acute hepatitis E. Sequence comparison revealed that the swine and human isolates of HEV share 97.3% identity. Phylogenetic analyses further showed that the Taiwan swine and human isolates of HEV form a distinct branch divergent from all other known strains of HEV, including the U.S. swine strain. To examine the potential risk of cross-species transmission of swine HEV to humans, the seroprevalences of anti-HEV IgG in 30 swine handlers, 20 pork dealers, and 50 control subjects were assessed and were found to be 26.7, 15, and 8%, respectively (for swine handlers versus controls,P= 0.048). Our findings may help provide an understanding of the modes of HEV transmission and may also raise potential public health concerns for HEV zoonosis.

We have developed a polymerase chain reaction (PCR) method for sequencing of tobacco chloroplast genome. In a mixture containing chloroplast DNA, 5'-end-labeled oligonucleotide primer, Taq DNA polymerase and reaction buffer, we were able to sequence a segment of chloroplast 16S rRNA gene. The results showed that the 750 bp of DNA sequenced were identical to the sequence reported, indicating that direct sequencing method that we have developed is useful for the sequencing of chloroplast genome. To analyze the chloroplast genome more rapidly in those in vitro grown plantlets, we also developed a simple method which is applicable for the amplifications and sequencing of chloroplast 16S rRNA fragment from either 0.15 g of tobacco leaf or stem tissue. The readable sequences obtained from the presented methods were consistent with the published sequence.

Single‐stranded conformation polymorphism (SSCP) analysis was used to examine the mutations of the chloroplast 16S rRNA locus of streptomycin‐resistant mutants in Nicotiana plumbaginifolia.DNA fragments of 121, 517, 968 and 1578 bp, each possessing a known point mutation, were generated by polymerase chain reaction (PCR). The resulting fragments were denatured and separated by nondenaturing polyacrylamide gel electrophoresis. Compared to the patterns of the wild‐type DNA fragments, the bands of the single‐stranded DNA fragments of 121 and 517 bp with base changes were shifted. However, no pattern variations were detected from the DNA fragments of 968 and 1578 bp generated from both wild‐type and mutants.

A total of 20Bacillus subtilis F29-3 mutants defective in fengycin biosynthesis was obtained by Tn917 mutagenesis. Cloning and mapping results showed that the transposon in these mutants was inserted in eleven different locations on the chromosome. We were able to use the chromosomal sequence adjacent to the transposon as a probe to screen for cosmid clones containing the fengycin biosynthesis genes. One of the clones obtained, pFC660, was 46 kb long. Eight transposon insertion sites were mapped within this plasmid. Among the eleven different mutants analyzed, four mutants had Tn917 inserted in regions which encoded peptide sequences similar to part of gramicidin S synthetase, surfactin synthetase, and tyrocidine synthetase. Our results suggest that fengycin is synthesized nonribosomally by the multienzyme thiotemplate mechanism.

A 1.6-kb DNA region required for the replication of pSW500 fromErwinia stewartii SW2 has been identified. DNA sequencing analysis revealed that this DNA fragment consists of a DnaA box, seven 16-bp direct repeats, and a 1005-bp open reading frame. The seven direct repeats have been demonstrated to mediate the incompatibility function of the plasmid. Primer extension analysis showed that the 1005-bp ORF is transcribed in vivo and the +1 site of the transcript is located 113 bp upstream from the translation initiation codon of the ORF. Complementation studies showed that this ORF is required for the replication of the plasmid and may encode a replication protein, RepA. Gene fusion studies revealed that the expression ofrepA is autoregulated by RepA. We also found that the pSW500 replicon has a copy number of approximately two and that the plasmid is stably maintained inEscherichia coli, thus demonstrating that the replicon contains all the elements required for copy number control and plasmid stability inE. coli. Curing of pSW500 fromE. stewartii SW2 revealed that loss of pSW500 did not have any obvious effect on morphology or physiology of the cells, suggesting that pSW500 does not encode a function that is indispensable for the survival of the organism.

Different mutations were generated in the diffused-form early antigen (EA-D) of Epstein-Barr virus (EBV) for study of the effects of these mutations in DNA binding, stimulating the activity of EBV-specific DNA polymerase (EBV-DP), and binding to monoclonal antibody R3. Results revealed that the N-terminal 303 amino acids were essential for DNA binding and were sufficient for the enhancement of the activity of EBV-specific DNA polymerase. Deletion study also showed that the region recognized by the R3 monoclonal antibody was located between aa 315 and aa 377. Our results failed to demonstrate the binding between EA-D and EBV-DP, using the proteins synthesized in vitro,suggesting that direct contact between the two proteins is not required for the EBV-DP activity in vitro.We have generated fusion between EA-D and DNA-binding domain of yeast GAL4 protein; however, this fusion protein was not able to transactivate the promoter containing UAS sequence in P3HR1 cells.

Theoriregion of anErwinia stewartiiplasmid, pSW1200 (106 kb), has been cloned and sequenced. This region consists of a gene encoding a protein which has 91% similarity and 73% identity with the RepA protein of bacteriophage P1. Theoriregion also consists of eight copies of 19-bp iterons which are highly homologous to the iterons of P1. Similar to plasmid P1, pSW1200 replicon has a copy number of approximately 1. On the other hand, the copy number increases about ninefold if three of the iterons located downstream fromrepAgene are deleted. We also demonstrate that pGEM-5Z consisting of a copy of P1 iteron is incompatible with a pSW1200 derivative, pSW1201, suggesting that pSW1200 and P1 DNA are incompatible and both belong to the IncY group.

The nucleotide sequence of pSW200 ofErwinia stewartiiSW2 was determined. This plasmid is 4367 bp long, consisting of four mobilization genes,mobCABD,and an origin of replication homologous to those of ColE1-type plasmids. The plasmid also contains a region of forty-one 15-bp repeats. Deleting this region does not affect the stability or the copy number when maintained as sole plasmid in the cell. However, the plasmid is rapidly lost when a homoplasmid with the intact repeat region is introduced into the cell. The function of this region may provide pSW200 an advantage in competing with an incompatible plasmid in the cell.

ABSTRACTThe BZLF1 gene of Epstein-Barr virus (EBV), which encodes a transcription factor, Zta, is transcribed into monocistronic and bicistronic mRNAs from two different promoters during the immediate-early stage of the EBV lytic cycle. It is generally accepted that the Zta protein translated from the monocistronic mRNA profoundly influences the activation of the EBV lytic cycle. In this study, we constructed a plasmid, pCMV-RZLUC, which can transcribe a bicistronic mRNA consisting of BRLF1 and a BZLF1-lucfusion gene under latent conditions. P3HR1 cells transfected with this plasmid produce a luciferase activity which is approximately 17-fold higher than the activity exhibited by pRZLUC, a plasmid incapable of transcribing the bicistronic mRNA. Genetic analyses indicated that mutations in BRLF1 not only can decrease the translation of the fusion gene from the bicistronic mRNA but can also be complemented by a functional BRLF1 gene in cis. This observation implies that the product of BRLF1, Rta, is involved in the translation of the downstream gene. Results presented herein also demonstrate that these mutations cannot be complemented in transwith a plasmid overexpressing Rta, suggesting that the amount of Rta in the vicinity of the intercistronic region may be crucial for the translation. Furthermore, our results correspond to those of previous investigations indicating that the Zta protein can be translated from the bicistronic mRNA and that, similar to the translation of bicistronic ZLUC, mutations in BRLF1 also hinder the translation of Zta from the BRLF1-BZLF1 bicistronic mRNA. Translation of Zta from the bicistronic mRNA may play an essential role in the activation of the EBV lytic cycle.

The carotenoid biosynthesis operon of Erwinia herbicola Eho13 consists of five genes, which are organized in the order crtE-crtX-crtY-crtI-crtB. These genes, with the exception of crtX, encode functions of ß-carotene biosynthesis and give an orange-coloured phenotype in Escherichia coli. Since crtX is not involved in the biosynthesis of ß-carotene, deletion of this gene does not alter the phenotype of pigmented cells. On the other hand, insertion of Tn1000 into crtX or into the upstream untranslated region of the operon resulted in a light-yellow, rather than an unpigmented phenotype, indicating that Tn1000 does not exert a strong polar effect when inserted in this operon. RNA analysis revealed that the sequence downstream from the insertion site was transcribed at a low level. Primer extension showed that the “-35”-like sequence in the terminal inverted repeats was not responsible for the transcription of the downstream sequence. Furthermore, primer extension and polymerase chain reaction (PCR) studies revealed that RNA transcribed from the promoters inside of Tn1000 was extended through the terminal inverted repeats into the adjacent sequences. In addition Tn1000, in either orientation, was able to generate fusion transcripts when placed upstream of a promoterless tetracycline-resistance gene and resulted in cells resistant to the drug. These results showed that Tn1000 insertion transcriptionally activates the DNA sequences adjacent to the transposon.

The nucleotide sequence of the smallest plasmid ofErwinia stewartiiSW2 was determined. This plasmid, pSW100 (4272 bp), consists of a 702-bp region homologous to the origins of replication of plasmids p15A, ColE1, and ColA. Plasmid pSW100 also contains sequences homologous to thebomregion andmobCABDgenes of ColE1, except that a single-base deletion inmobAhas been detected. This deletion did not affect the mobilization ability of pSW100 by an endogenous conjugative plasmid ofE. stewartiiSW2, pDC250. Plasmid pSW100 also has a 1596-bp open reading frame with five 132-bp perfect repeats. However, this open reading frame and the repeats are not required for plasmid replication. Furthermore, curing of pSW100 did not cause any phenotypic change, suggesting that this plasmid is not essential for the survival of the organism.

Two types of expression vectors containing thePtacorPLpromoters, synthetic ribosomalbinding sequences (RBS), and a transciptional terminator (rrnBT1T2) were constructed for the production of porcine growth hormone (pGH) inEscherichia coli. The results demonstrated that the nucleotide sequence between the RBS and the start codon, ATG, was critical for the yields of pGH. The rrnBT1T2terminator was essential for the efficient expression ofpGHin thePtac-driven vectors, but reduced the synthesis of pGH in thePL-vectors. A leaky repression was observed in the JM103 cells containing thePtac-plasmids.

Epstein-Barr virus is known to cause nasopharyngeal carcinoma. Although oral cavity is located close to the nasal pharynx, the pathogenetic role of Epstein-Barr virus (EBV) in oral cancers is unclear. This molecular epidemiology study uses EBV genomic microarray (EBV-chip) to simultaneously detect the prevalent rate and viral gene expression patterns in 57 oral squamous cell carcinoma biopsies (OSCC) collected from patients in Taiwan. The majority of the specimens (82.5%) were EBV-positive that probably expressed coincidently the genes for EBNAs, LMP2A and 2B, and certain structural proteins. Importantly, the genes fabricated at the spots 61 (BBRF1, BBRF2, and BBRF3) and 68 (BDLF4 and BDRF1) on EBV-chip were actively expressed in a significantly greater number of OSCC exhibiting exophytic morphology or ulceration than those tissues with deep invasive lesions ( and .0141, resp.). The results may thus provide the lead information for understanding the role of EBV in oral cancer pathogenesis.